Biocatalysts are recognized as key materials for “sustainable economic development”, including clean technology for the production of energy, industrial materials, etc, and various efforts have been made to screen enzymes having new chemical reactivity, specificity, and stability. Methods for high-throughput screening of antibiotic resistance genes, enzymes essential for growth, several industrial enzymes (e.g., amylase, lipase, protease, etc.), and the like on solid media have been known, but most enzymatic functions depend on the technology for analysis of individual activity that requires much time and cost.
In recent years, studies on the application of directed evolution technology to secure new genetic resources from microbial genomes or metagenomes or improve the activity of existing genes to develop highly useful biocatalysts have emerged as important strategies in bioengineering technology. Thus, there is a need to develop novel high-sensitivity screening technology for high-throughput detection of the activities of very small amounts of enzymes.
Methods of obtaining new enzyme genes from various genetic resources, such as microbial genomes and environmental DNA (metagenome), include a sequence-based screening method that comprises sequencing DNA, performing a PCR reaction, and obtaining the amplified gene. Utilizability of this method is increasing day by day as genome information increases rapidly, and it has an advantage in that only desired genes can be specifically selected. However, this method has a shortcoming in that, because it can be applied only when accurate information about the nucleotide sequences of target genes is known, the application thereof is limited to a portion of genetic resources.
In addition, a function-based screening method that selects genes based on gene function, that is, enzyme activity, is also widely used. To screen enzyme activity by this method, methods of isolating microorganisms directly from environmental samples, including samples from soil, rivers, industrial wastewater, seawater, and forests, have been mainly used. However, the amount of microbial species that can actually be cultured in laboratories is as small as less than 1% of microorganisms present in nature. In recent years, a strategy of constructing genetic resources as metagenomic libraries by isolating DNA directly from environmental samples without culturing microorganisms has been actively attempted. Thus, there is a rising interest on developing a screening technology for detecting industrially useful enzymatic activities directly from metagenomic libraries.
Meanwhile, main technologies for high-throughput analysis of enzymatic activities include: 1) automated multiplex assay technology utilizing well plates; 2) a method of observing color development or a clear zone (halo) on solid medium; and 3) a selective isolation method utilizing nutritionally deficient microorganisms. These methods are based on the actual activity of an enzyme, and thus have an advantage in that they can precisely select a gene of desired function. However, because each detection technology is required for each enzymatic activity, the general use of these methods is limited. Additionally, the effects of these methods are further reduced when the transcription, translation, or expression of a foreign gene in host cells is low or problems such as protein folding or secretion arise. Indeed, in the case of new enzymes derived from genetic resources, such as new microbial genomes and metagenomes, which have high genetic diversity and the genetic characteristics of which is unknown, their expression levels in recombinant microorganisms is very low, and thus it is very difficult to apply the above high-throughput assay method to these enzymes. Thus, there has been a continual need to develop a new high-throughput screening principle according to which even the activity of an enzyme that is expressed at a very low level can be detected with high sensitivity.
In this context, a study on artificial genetic circuitry of detecting the activity of enzymes such as intracellular protease by genetic engineering technology based on the principle of transcriptional activation in a yeast 3-hybrid system was reported and received attention. In addition, technology of detecting enzymatic activities using products resulting from the action of foreign enzymes as nutrients for cells or detecting the enzymatic activities of foreign genes in recombinant E. coli by introducing the recombinant E. coli with transcription regulatory proteins from other microorganisms, by redesigning microbial metabolic pathways, is being actively studied. In addition, efforts to develop protein engineering technology for modifying the substrate specificity of regulatory proteins are being actively made. For example, modifying the substrate specificity of the regulatory protein HbpR, which binds to 2-hydroxybiphenyl (2-HBP), so as to specifically recognize 2-chlorobiphenyl (2-CBP) having chloro- in place of hydroxy-, was also studied (Beggah et al., (2008) Microb. Biotechnol. 1(1): 68-78).
Thus, detecting the products of enzymatic reactions using regulatory proteins can be used as innovative technologies for screening new enzymes. However, such technologies are merely technologies for screening a small number of specific enzymatic activities for which substrate products and regulatory proteins are elucidated, and these technologies cannot be universal systems that can be applied to various enzymatic activities.
Other technologies for screening new enzymes include SIGEX (substrate-induced gene expression screening) reported by the Watanabe research group in 2005. This technology is based on screening promoters whose transcriptional activity is induced by added substrates. Thus, it is not a technology for directly screening enzymatic activity, but is a technology for indirectly detecting genes. Namely, enzymatic functions which are not associated with transcriptional activation are not detected by this technology. In this technology, there is an advantage in that, because FACS analysis can be used, large amounts of samples can be treated within a short time. However, this technology is difficult to apply to a metagenomic library containing large genes having a size of 20-30 kb, and GFP activation in the direction of genes in a library cannot appear (Uchiyama et al., (2005) Nat. Biotech. 23: 88-93).
Meanwhile, studies on a technology for detecting phenolic compounds have been Known (Korean Patent Registration No. 10-0464068), but a study on the use of this technology to detect enzymatic activities has not yet been reported before.
The present inventors have conducted studies on a method capable of detecting various enzymatic activities. As a result, based on the fact that various phenol-release compounds capable of liberating phenol can be used as substrates in many enzymatic reactions, the present inventors have constructed artificial genetic circuits detecting phenols and found that the use of the artificial genetic circuits allows the measurement of quantitative activities of reporter genes, such as fluorescent reporter genes and antibiotic resistance genes, the expression of which was induced. Thus, the present inventors have confirmed the effectiveness and general utility of this technology by collecting and isolating genes having tyrosine phenol-lyase and alkaline phosphatase enzyme activities from a metagenomic library by high-throughput screening (million/day) using such genetic circuits, thereby completing the present invention.